Gram-negative bacteria Yersinia pestis (Yp), Francisella tularensis (Ft) and Burkholderia pseudomallei (Bp) are potential agents of a bioterror attack. This grant proposes to utilize large-scale quantitative proteomic technology to analyze the whole cell protein of Yp, Ft and Bp grown under environmental conditions known to induce expression of bacterial virulence factors. Proteomic technology will also be applied to analysis of regulated outer membrane and secreted proteins, which often contribute to the pathogenesis of Gram-negative infections. In addition, since intracellular lifestyle is likely essential for the pathogenesis of Ft and Bp, technology will be developed to analyze protein expression of these bacteria within the macrophage. Results of proteomic analysis will be integrated with results of transcriptional profiling of Yp, Ft and Bp. This global postgenomic analysis should provide a comprehensive overview of the regulatory networks that mediate virulence properties of these bacteria. Finally, our results should help define components for the development of protective vaccines and antimicrobial therapies.